In the field of biomedical imaging, various methods of optical imaging of biomedical tissues known in the art offer significant advantages over other biomedical imaging approaches. These advantages include non-ionizing radiation, wide range of resolution, numerous and effective contrast mechanisms, and relatively compact and inexpensive instrumentation. Optical imaging can be applied to a wide range of biological systems, from cells and subcellular organelles, in vivo and ex vivo tissues, to organs and whole body of a subject, covering the length scales of nanometers and micrometers to millimeters and centimeters. The contrast mechanisms include absorption, transmission, polarization, fluorescence, phase interference, nonlinear and multiphoton processes, as well as time behavior of these processes. Another very important dimension in optical imaging is the effect of these processes as functions of the wavelength of the light—i.e. the spectroscopy of the images. Through spectral imaging, one can monitor for detailed biochemical and biomedical parameters of the specimen.
Optical microscopy and its applications in biomedical imaging have been experiencing a remarkable growth over past few decades, thanks to such technological developments as lasers and digital acquisition and processing of images. One of the main thrust areas of the development of modern microscopy is three-dimensional microscopy, where one acquires a three-dimensional image with every image plane sharply in focus. This is in contrast to conventional microscopy where the image of the in-focus plane is superposed with a blurred image of out-of-focus planes.
In confocal scanning microscopy, CSM, the out-of-focus signal is spatially filtered out by confocal aperturing of the object illumination and the detector points. The three-dimensional image is constructed by pixel-by-pixel mechanical scanning of the entire object volume, which places a fundamental limit on the image acquisition speed.
Another more recent development in 3D microscopy is optical coherence tomography (OCT), where the axial resolution of a few μm is provided by interferometric measurement of the time-of-flight of short-coherence light. In a typical arrangement, a Michelson-type interferometer is illuminated by femtosecond laser or superluminescent LED, and the reference arm is dithered to generate a heterodyne signal in the interference with the back-scattered light from the sample point. The two- or three-dimensional image is constructed from the mechanical scanning over the sample area or volume, as in confocal scanning microscopy. In order to maintain the high speed of the z-scan, a considerably large depth of field, approximately a mm, is needed, which compromises the lateral resolution to a few μm. It is known in the art to sue a confocal adaptation to improve the resolution. The heterodyne detection allows for very high sensitivity and unique capabilities such as Doppler velocity detection of blood flow. OCT-based imaging systems are being developed for diverse areas of medical imaging including retinal structures, endoscopy of gastrointestinal tract and catheter-based intravascular imaging. As a coherent imaging technique, the OCT is capable of penetrating a larger distance into highly scattering media such as biological tissues and ceramics.
Scanning microscopies, including the confocal microscopy and optical coherence tomography, have a number of distinct advantages such as relaxed requirements on the imaging optics and high sensitivity and high resolution. On the other hand, the mechanical scanning is a major limiting factor in the image acquisition speed. Parallel acquisition of two-dimensional images while maintaining the optical sectioning characteristics of CSM or OCT would have obvious advantages. In CSM, such 2D imaging is approximated with a large number of well-spaced apertures, such as rotating Nipkow disk or multi-aperture scanning using an electro-optic spatial light modulator, but the light efficiency or image contrast tends to be low. Wide field optical sectioning is also achieved by structured light microscopy where a moving grating pattern illuminates the object and processing of several images extracts the in-focus sectioned image.
With OCT, it is known that full-field interferometric images can be acquired using broadband light sources, and the regions of the image that do contain interferometric information can be extracted by digital processing of the CCD images, thus generating optical section images variously known as wide-field, full-field, or two-dimensional OCT. The 3D image is constructed by mechanical scanning of the axial direction only. In a simple 2DOCT system, the light source illuminates the entire area of object to be imaged and its interference with the reference beam is imaged using a CCD array. For example, the reference optical path length is modulated by half wavelength, and pair of images is taken with a □ phase shift between them. The difference image then highlights the areas of interference within an axial depth equal to the coherence length, while the rest of the image area is significantly attenuated. A quasi-lock-in image acquisition is known in the art demonstrating synchronous illumination, instead of synchronous detection, due to the limited frame rate of the CCD camera, 30 Hz. High-frequency (50 kHz) true lock-in image acquisition has also been demonstrated in the art using custom made smart array detector, although with a limited number of pixels (58×58).
As such, the prior art demonstrates the potential for high-speed high-resolution 3D microscopy with very respectable sensitivity or dynamic range, at least ˜80 db, and promises to have significant impact on OCT applications where acquisition speed is critical, as in real-time in-vivo ophthalmic and endoscopic imaging.
Unlike conventional microscopy, most of the current developments of 3D microscopy usually discard the natural color information of the object, whereas in some of the critical application areas of OCT such as ophthalmic and dermatological imaging, the color and texture of relatively thin top layers of the tissues can provide vital information in a format that is familiar to medical specialists in these areas.
Accordingly, what is needed in the art is a full-color 3D microscopic imaging system and method utilizing wide-field optical coherence tomography resulting in a 3D image with full natural color representation.
However, in view of the prior art considered as a whole at the time the present invention was made, it was not obvious to those of ordinary skill in the pertinent art how the identified need could be fulfilled.